Application of chlorogenic acid and resveratrol in improving growth performance and / or disease resistance of procambarus clarkia

Abstract

The invention provides application of chlorogenic acid and resveratrol in improving the growth performance and / or disease resistance of procambarus clarkii, and belongs to the field of aquatic nutrition. It is found that the chlorogenic acid and the resveratrol are jointly used according to the mass ratio of (1-3): (1-3), the specific growth rate and the survival rate of the procambarus clarkia can be remarkably increased, and the white spot syndrome virus resisting capacity of the procambarus clarkia is improved. The invention further provides a feed additive for improving the growth performance and the disease resistance of the procambarus clarkii and a procambarus clarkii feed, and the health level and the economic benefits of the procambarus clarkii can be comprehensively improved.

Description

Technical Field

[0001] The invention belongs to the field of aquatic nutrition, and specifically relates to the application of chlorogenic acid combined with resveratrol in improving the growth performance and / or disease resistance of Procambarus clarkii. Background Art

[0002] Procambarus clarkii (Latin name: Procambarus clarkii) is an animal of the genus Procambarus in the family Crayfish. Procambarus clarkii has a high protein content, is rich in magnesium, zinc, iodine, selenium, astaxanthin, etc., and has soft meat and is easy to digest. It can protect the cardiovascular system, reduce the cholesterol content in the blood, prevent arteriosclerosis, and dilate the coronary arteries, which is beneficial for preventing hypertension and myocardial infarction. It is deeply loved by people. However, with the continuous expansion of the scale of Procambarus clarkii aquaculture, the continuous increase in aquaculture density, and the continuous deterioration of the aquaculture ecological environment, its adaptability and resistance have been greatly affected. Disease problems have become increasingly prominent during the aquaculture process. The slightest carelessness can cause the occurrence and spread of diseases, which can affect feeding and slow growth at the mildest, and cause a large number of deaths at the worst, causing huge economic losses. There are many types of viruses that cause Procambarus clarkii diseases, among which white spot syndrome is the most serious, which seriously threatens the healthy and sustainable development of Procambarus clarkii aquaculture.

[0003] The disease outbreak of Procambarus clarkii is not caused by a single factor, but the result of the combined effects of the destruction of the breeding environment and the weakening of the shrimp's disease resistance. Procambarus clarkii is an invertebrate with only a non-specific immune system, and the traditional vaccine method of preventing and treating diseases is not effective. Although the use of feed additives to regulate the immune ability of Procambarus clarkii in oral form is an effective method. However, there are not many feed additives that can effectively improve the disease resistance of Procambarus clarkii, and most of them are concentrated in Chinese herbal medicine preparations, etc., but the composition of this type of substance is complex, the dosage is not easy to determine, and the application effect is not easy to grasp. Therefore, it is very necessary to develop efficient and practical feed additives and clarify the use methods to improve the disease resistance of Procambarus clarkii.

[0004] The biological functions of chlorogenic acid (CGA) and resveratrol (RES) have been partially reported in animal production, but they are mostly concentrated on livestock and poultry. There are relatively few studies on aquatic animals, and even fewer studies on farmed shrimp. So far, only a few studies have shown that chlorogenic acid and resveratrol are added to the feed of Penaeus vannamei. However, Penaeus vannamei belongs to the order Decapoda of crustaceans and is a small marine arthropod. Procambarus clarkii belongs to the subphylum Procrustacea and usually lives in freshwater lakes, rivers and ponds. There are significant differences between the two in many aspects, including appearance, growth environment, living habits, feeding habits and nutritional value. The application effects of the same substance in Penaeus vannamei and Procambarus clarkii are significantly different, and the nutritional selection in Penaeus vannamei feed is not suitable for Procambarus clarkii. The application of chlorogenic acid and resveratrol in Procambarus clarkii feed has not been reported. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the object of the present invention is to provide the use of chlorogenic acid combined with resveratrol in improving the growth performance and / or disease resistance of Procambarus clarkii. Chlorogenic acid and resveratrol have a synergistic effect, which can significantly improve the specific growth rate and survival rate of Procambarus clarkii, and improve the ability of Procambarus clarkii to resist white spot syndrome virus (WSSV).

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of chlorogenic acid combined with resveratrol in the preparation of a product for improving the growth performance and / or disease resistance of Procambarus clarkii, wherein the mass ratio of the chlorogenic acid to resveratrol is (1-3):(1-3).

[0008] The present invention also provides a feed additive for improving the growth performance and / or disease resistance of Procambarus clarkii, wherein the feed additive is composed of chlorogenic acid and resveratrol in a mass ratio of (1-3):(1-3).

[0009] The present invention also provides the use of the feed additive in the cultivation of Procambarus clarkii, wherein the addition amount of the feed additive accounts for 0.04% to 0.16% of the total mass of the feed.

[0010] Preferably, the feed additive increases the specific growth rate and survival rate of Procambarus clarkii.

[0011] Preferably, the feed additive improves the ability of Procambarus clarkii to resist white spot syndrome virus.

[0012] Preferably, the feed additive increases the activities of lysozyme and phenoloxidase in the serum of Procambarus clarkii and reduces the MDA content in the serum of Procambarus clarkii.

[0013] The present invention also provides a feed for Procambarus clarkii, which comprises, by weight, 50-150 parts of fish meal, 100-200 parts of soybean meal, 40-80 parts of cottonseed protein, 60-150 parts of rapeseed meal, 50-200 parts of peanut meal, 30-50 parts of corn DDGS, 100-280 parts of wheat flour, 5-20 parts of sodium alginate, 15-25 parts of calcium dihydrogen phosphate, 5-30 parts of yeast powder, 5-30 parts of soybean oil, 5-30 parts of fish oil, 5-15 parts of soybean lecithin oil, 2-8 parts of cholesterol, 5-20 parts of multivitamin, 2-8 parts of sodium chloride, 1-2.5 parts of choline chloride, 1-2.5 parts of vitamin C, 0.5-2 parts of chitosan, 10-50 parts of bentonite and 0.4-2 parts of feed additives; the feed additives are the above-mentioned feed additives.

[0014] Preferably, the multivitamin includes 5-10 mg / kg thiamine, 5-10 mg / kg riboflavin, 1000-5000 IU / kg vitamin A, 3500-1000 IU / kg vitamin D, 20-50 mg / kg vitamin E, 35-10 mg / kg vitamin K, 5-10 mg / kg pyridoxine, 0.05-0.2 mg / kg biotin, 0.02-0.05 mg / kg cyanocobalamin, 5-20 mg / kg calcium pantothenate, 0.5-1 mg / kg folic acid, 20-50 mg / kg niacin, vitamin C. 50-200mg / kg, ferrous sulfate 200-400mg / kg, potassium iodate 0.5-1.5mg / kg, cupric chlorohydroxide 2-5mg / kg, zinc sulfate 50-150mg / kg, manganese sulfate 10-50mg / kg, sodium selenite 0.2-0.6mg / kg and cobalt chloride hexahydrate 0.5-1mg / kg.

[0015] The present invention also provides a method for preparing the above-mentioned Procambarus clarkii feed, comprising the following steps: uniformly mixing fish meal, soybean meal, cottonseed protein, rapeseed meal, peanut meal, corn DDGS, wheat flour, sodium alginate, calcium dihydrogen phosphate, yeast powder, cholesterol, multivitamin, sodium chloride, choline chloride, vitamin C, chitosan, bentonite and feed additives, then adding fish oil, corn oil and soybean lecithin, granulating and drying.

[0016] The present invention also provides the use of the Procambarus clarkii feed in preparing a product for improving the growth performance and / or disease resistance of Procambarus clarkii.

[0017] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0018] The present invention provides the use of chlorogenic acid combined with resveratrol in improving the growth performance and / or disease resistance of Procambarus clarkii. The present invention finds that chlorogenic acid and resveratrol have a synergistic effect, can significantly improve the specific growth rate and survival rate of Procambarus clarkii, and improve the ability of Procambarus clarkii to resist white spot syndrome virus.

[0019] The present invention utilizes chlorogenic acid and resveratrol for proportioning, and chlorogenic acid and resveratrol are composed according to a specific ratio to obtain a feed additive for Procambarus clarkii, and the feed additive / feed is further used to prepare Procambarus clarkii feed. The obtained feed additive / feed can significantly improve the growth performance of Procambarus clarkii, especially the specific growth rate and survival rate of Procambarus clarkii, can improve the ability of Procambarus clarkii to resist white spot syndrome virus, significantly improve the survival rate of Procambarus clarkii after infection with WSSV, increase the activities of lysozyme and phenoloxidase in the serum of Procambarus clarkii, reduce the MDA content in the serum of Procambarus clarkii, and help to comprehensively improve the health level and economic benefits of Procambarus clarkii. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :Effects of chlorogenic acid and resveratrol added to feed on the resistance of Procambarus clarkii to WSSV infection;

[0021] Figure 2 :Effects of chlorogenic acid and resveratrol added to feed on nonspecific immune parameters of Procambarus clarkii. DETAILED DESCRIPTION

[0022] The present invention provides the use of chlorogenic acid combined with resveratrol in the preparation of a product that improves the growth performance and / or disease resistance of Procambarus clarkii, wherein the mass ratio of chlorogenic acid to resveratrol is (1-3):(1-3), preferably 1:3. Chlorogenic acid is a conjugated phenolic acid that has the effects of anti-oxidation, anti-inflammatory, antibacterial, anti-tumor, regulating sugar metabolism and lipid metabolism, protecting the liver and intestines, and protecting the nervous system. Resveratrol is a non-flavonoid polyphenol compound with a stilbene structure, which has multiple biological functions such as antibacterial, anti-inflammatory, antioxidant, regulating sugar and lipid metabolism. The present invention finds that chlorogenic acid and resveratrol have a synergistic effect when used in combination in a specific ratio, which can significantly improve the specific growth rate and survival rate of Procambarus clarkii, and improve the ability of Procambarus clarkii to resist white spot syndrome virus.

[0023] The present invention also provides a feed additive for improving the growth performance and / or disease resistance of Procambarus clarkii, wherein the feed additive is composed of chlorogenic acid and resveratrol in a mass ratio of 1:3. The present invention obtains a feed additive for Procambarus clarkii by combining chlorogenic acid and resveratrol in a specific ratio, wherein the addition amount of the feed additive accounts for 0.04% to 0.16% of the total mass of the feed, and the total mass of the feed includes the basic feed and the feed additive. The feed additive of the present invention can improve the specific growth rate and survival rate of Procambarus clarkii, can improve the ability of Procambarus clarkii to resist white spot syndrome virus, significantly improve the survival rate of Procambarus clarkii after infection with WSSV, improve the activity of lysozyme and phenoloxidase in the serum of Procambarus clarkii, reduce the MDA content in the serum of Procambarus clarkii, and help to comprehensively improve the health level and economic benefits of Procambarus clarkii.

[0024] The present invention also provides a feed for Procambarus clarkii, which comprises, by weight, 50 to 150 parts of fish meal, 100 to 200 parts of soybean meal, 40 to 80 parts of cottonseed protein, 60 to 150 parts of rapeseed meal, 50 to 200 parts of peanut meal, and corn DDGS. 30-50 parts, wheat flour 100-280 parts, sodium alginate 5-20 parts, calcium dihydrogen phosphate 15-25 parts, yeast powder 5-30 parts, soybean oil 5-30 parts, fish oil 5-30 parts, soybean lecithin oil 5-15 parts, cholesterol 2-8 parts, multivitamin 5-20 parts, sodium chloride 2-8 parts, choline chloride 1-2.5 parts, vitamin C 1-2.5 parts, chitosan 0.5-2 parts, bentonite 10-50 parts, feed additives 0.4-2 parts; preferably including 50 parts of fish meal, 150 parts of soybean meal, 60 parts of cottonseed protein, 130 parts of rapeseed meal, 100 parts of peanut meal, corn DDGS 50 parts, wheat flour 280 parts, sodium alginate 20 parts, calcium dihydrogen phosphate 25 parts, yeast powder 20 parts, soybean oil 20 parts, fish oil 15 parts, soybean lecithin oil 15 parts, cholesterol 5 parts, multivitamin 10 parts, sodium chloride 5 parts, choline chloride 2.5 parts, vitamin C 2.5 parts, chitosan 1 part, bentonite 37.4 parts, feed additives 1.6 parts; the feed additives are the above-mentioned feed additives.

[0025] The multivitamin of the present invention comprises 5-10 mg / kg of thiamine, 5-10 mg / kg of riboflavin, 1000-5000 IU / kg of vitamin A, 3500-1000 IU / kg of vitamin D, 20-50 mg / kg of vitamin E, 35-10 mg / kg of vitamin K, 5-10 mg / kg of pyridoxine, 0.05-0.2 mg / kg of biotin, 0.02-0.05 mg / kg of cyanocobalamin, 5-20 mg / kg of calcium pantothenate, 0.5-1 mg / kg of folic acid, 20-50 mg / kg of niacin, and vitamin C. 50-200mg / kg, ferrous sulfate 200-400mg / kg, potassium iodate 0.5-1.5mg / kg, copper chlorohydroxide 2-5mg / kg, zinc sulfate 50-150mg / kg, manganese sulfate 10-50mg / kg, sodium selenite 0.2-0.6mg / kg and cobalt chloride hexahydrate 0.5-1mg / kg; preferably including thiamine 5mg / kg, riboflavin 10mg / kg, vitamin A 5000IU / kg, vitamin D3 1000 IU / kg, vitamin E 40mg / kg, vitamin K3 10mg / kg, pyridoxine 10mg / kg, biotin 0.1mg / kg, cyanocobalamin 0.02mg / kg, calcium pantothenate 20mg / kg, folic acid 1mg / kg, niacin 40mg / kg, vitamin C 150mg / kg, ferrous sulfate 303mg / kg, potassium iodate 1.3mg / kg, cupric chlorohydroxide 5mg / kg, zinc sulfate 138mg / kg, manganese sulfate 36mg / kg, sodium selenite 0.6mg / kg and cobalt chloride hexahydrate 0.8mg / kg.

[0026] The present invention also provides a method for preparing the above-mentioned crayfish feed, comprising the following steps: fish meal, soybean meal, cottonseed protein, rapeseed meal, peanut meal, corn DDGS, wheat flour, sodium alginate, calcium dihydrogen phosphate, yeast powder, cholesterol, multivitamin, sodium chloride, choline chloride, vitamin C, chitosan, bentonite, and feed additives are mixed evenly, and then fish oil, corn oil and soybean lecithin are added, granulated, and dried. The raw material mixing is preferably that each raw material is gradually mixed and then thoroughly mixed using a V-type mixer; the granulation is preferably that a small amount of water is added and then the feed particles are extruded through a twin-screw extruder; the drying is preferably baked at 75°C for 20 minutes, and then naturally air-dried. The feed prepared by the present invention is stored at -20°C.

[0027] The present invention also provides the use of the Procambarus clarkii feed in preparing a product for improving the growth performance and / or disease resistance of Procambarus clarkii.

[0028] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] In the specific examples of the present invention, chlorogenic acid was purchased from Shaanxi Yuzhou Biotechnology Co., Ltd., and resveratrol was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. All kits in the specific examples of the present invention were from Shanghai ELISA Biotechnology Co., Ltd., and the relevant operations were strictly performed in accordance with the manual.

[0030] In the specific embodiment of the present invention, all data analyses were performed in GraphPadPrism software, and the results were expressed as mean ± standard deviation (mean ± SD). The differences between the groups were analyzed by one-way analysis of variance (ANOVA), and multiple comparisons were performed using Tukey; the results of the challenge experiment were analyzed for survival rate using Log-rank Kaplan-Meier analysis. All data results were considered to be significantly different when P < 0.05.

[0031] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0032] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0033] Example 1

[0034] 1. Experimental design

[0035] The basic feed was used as the control, and chlorogenic acid and resveratrol were added to the basic feed at a content of 400 mg / kg as a control. Chlorogenic acid and resveratrol were added at a mass ratio of 1:1, 1:2, 2:1, 1:3 and 3:1, respectively, to prepare 8 kinds of isonitrogen and isoenergetic feeds. The basic feed control group was recorded as CON, the basic feed with chlorogenic acid added alone was recorded as CGA, the basic feed with resveratrol added alone was recorded as RES, and the experimental groups with chlorogenic acid and resveratrol added at a mass ratio of 1:1, 1:2, 2:1, 1:3 and 3:1 were recorded as CR11, CR12, CR21, CR13 and CR31, respectively. The feed formula and nutritional composition of each group are shown in Table 1. The 8 groups of feeds were fed to Procambarus clarkii, and a 6-week breeding experiment was carried out to compare the changes of chlorogenic acid and resveratrol in different ratios on the growth performance, disease resistance and immune indicators of Procambarus clarkii.

[0036] Table 1 Feed formula and nutritional composition of each group

[0037]

[0038]

[0039] The multivitamin is: thiamine 5mg / kg, riboflavin 10mg / kg, vitamin A 5000IU / kg, vitamin D31000IU / kg, vitamin E 40mg / kg, vitamin K310mg / kg, pyridoxine 10mg / kg, biotin 0.1mg / kg, cyanocobalamin 0.02mg / kg, calcium pantothenate 20mg / kg, folic acid 1mg / kg, niacin 40mg / kg, vitamin C 150mg / kg, ferrous sulfate 303mg / kg, potassium iodate 1.3mg / kg, cupric chlorohydroxide 5mg / kg, zinc sulfate 138mg / kg, manganese sulfate 36mg / kg, sodium selenite 0.6mg / kg and cobalt chloride hexahydrate 0.8mg / kg.

[0040] The preparation method of the above-mentioned crayfish feed is as follows: all raw materials are first crushed and screened, and fish meal, soybean meal, cottonseed protein, rapeseed meal, peanut meal, corn DDGS, wheat flour, sodium alginate, calcium dihydrogen phosphate, yeast powder, cholesterol, multivitamin, sodium chloride, choline chloride, vitamin C, chitosan, bentonite, and feed additives are gradually mixed, and then thoroughly mixed using a V-type mixer (JS-14S, Zhejiang Chint Electric Co., Ltd., Zhejiang, China). Fish oil, corn oil and soybean lecithin are added to the mixture, a little water is added, and then feed pellets are extruded through a twin-screw extruder (M-256, South China University of Technology, Guangzhou, China). The feed pellets are baked at 75°C for 20 minutes, naturally air-dried and stored at -20°C.

[0041] 2. Feeding and management

[0042] 720 crayfish with an initial body weight of 4.82±0.21g were selected and divided into 8 treatments and fed with 8 kinds of feeds. Each treatment was repeated three times, with 30 shrimps in each repeat. The experiment was cultured in a 2m (length) × 1.5m (width) × 0.7m (height) canvas pond, and a 50cm × 25cm pseudo-nest with an aperture of 6.5cm was selected as a cover for crayfish to climb and hide. Fasting for 24h before and after the start of the experiment. At the beginning of the formal experiment, 5% of the shrimp body weight was fed at a fixed point twice a day (7:00am and 9:00pm), and the amount was increased or decreased according to the feeding situation; the sewage was cleaned once a day (7:00am), and the residual bait, dead shrimps and shrimp shells were removed. During the experiment, the feeding status and health status of the crayfish were observed, the water quality was checked regularly, the ammonia nitrogen content was monitored once a week by the bromate method, and the dissolved oxygen was monitored once a week by the Winkler titration method. During the experiment, the water temperature was (25±2)℃, and the pH value, ammonia nitrogen ratio and dissolved oxygen content were all at normal levels.

[0043] 3. Sample collection and growth performance analysis

[0044] After the 6-week rearing period, the crayfish were starved for 24 hours, and the survival rate (SR), final body weight (FBW), weight gain rate (WGR), specific growth rate (SGR) and feed conversion ratio (FCR) of each group of crayfish were evaluated. The results are shown in Table 2.

[0045] Survival rate (SR, %) = 100 × (final number of shrimps / initial number of shrimps).

[0046] Weight gain rate (WGR, %) = 100 × (final body weight - initial body weight) / initial body weight.

[0047] Specific growth rate (SGR, %) = 100 × [ln (final body weight - ln (initial body weight)] / day.

[0048] Feed conversion ratio (FCR) = feed intake / (final body weight - initial body weight).

[0049] Table 2 Effects of adding chlorogenic acid and resveratrol to feed on the growth performance of Procambarus clarkii

[0050]

[0051] The results showed that compared with the control group, the addition of chlorogenic acid and resveratrol alone or in combination in the feed could increase the terminal weight gain rate and specific growth rate of Procambarus clarkii. Compared with the addition of chlorogenic acid alone, the survival rate of Procambarus clarkii was significantly increased when the ratio of chlorogenic acid and resveratrol was 1:2, 2:1 and 1:3 in the feed, and the survival rate of Procambarus clarkii was significantly increased when the ratio of chlorogenic acid and resveratrol was 1:3 in the feed compared with the addition of resveratrol alone; compared with the addition of chlorogenic acid or resveratrol alone, the terminal weight of Procambarus clarkii was significantly increased when the ratio of chlorogenic acid and resveratrol was 1:2, 2:1, 1:3 and 3:1 in the feed, and the weight gain rate of Procambarus clarkii was significantly increased when the addition ratio was 1:2, 2:1 and 1:3; the specific growth rate of Procambarus clarkii was significantly increased when the addition ratio was 1:3. The above results show that adding chlorogenic acid and resveratrol alone or in mixture can improve the growth performance of Procambarus clarkii. Adding chlorogenic acid and resveratrol in a certain proportion is more conducive to improving the growth performance of Procambarus clarkii than adding them alone. The effect is most obvious when the addition ratio of chlorogenic acid and resveratrol is 1:3.

[0052] 4. Virus attack experiment

[0053] 1 g of shrimp muscle tissue containing WSSV virus was cut and added with 10 mL PBS. The tissue was ground with a glass tissue grinder in an ice bath and filtered with a 0.45 μm microporous membrane at 4 °C to prepare a 5 × 10 5 / μL of white spot syndrome virus suspension. After the 6-week feeding period, the challenge experiment was carried out. Thirty crayfish were randomly selected from each treatment group, and 50μL of WSSV virus suspension was injected into the shrimp muscle. The survival rate was monitored and recorded every 4 hours within 72 hours. The Mantel-Cox (log-rankχ 2 The difference between the two groups was analyzed using the test method.

[0054] The results are as follows Figure 1 As shown in the results, the addition of resveratrol alone or chlorogenic acid and resveratrol in the feed can significantly improve the survival rate of Procambarus clarkii after infection with WSSV. After Procambarus clarkii was infected with WSSV, the survival rate of Procambarus clarkii in the CR12, CR31 and CR13 groups was significantly higher than that in the CGA and RES groups (P < 0.05), among which the final survival rate of Procambarus clarkii in the CR13 group was the highest, followed by the CR12 group, and the CR31 group was the third. The survival rates of Procambarus clarkii in the CR11 and CR21 groups were higher than those in the CGA and RES groups, but there was no significant difference. The survival rate of Procambarus clarkii in the CR13 group was significantly higher than that in the CR11 and CR21 groups. The above results show that in terms of improving the disease resistance of Procambarus clarkii, the effect of adding chlorogenic acid and resveratrol in a ratio of 1:2, 3:1 and 1:3 is more obvious than that of adding chlorogenic acid or resveratrol alone, and the best effect is achieved when the addition ratio is 1:3.

[0055] 5. Immune index detection

[0056] After the 6-week rearing period, the crayfish were starved for 24 hours, and the hemolymph of 9 shrimps was randomly collected from each culture pond. The hemolymph from every 3 shrimps was used as a sample for the analysis of immune-related indicators. The collected samples were quickly frozen in liquid nitrogen and stored at -80°C for subsequent analysis. The hemolymph was naturally coagulated for 10-20 minutes at room temperature and centrifuged for 20 minutes (2000-3000r / min). The supernatant was carefully collected to detect the activities of lysozyme (LZM) and phenoloxidase (PO) and the content of malondialdehyde (MDA), which were determined using enzyme-linked immunosorbent assay (ELISA) kits ml556394, ml695266 and ml555268, respectively.

[0057] The results are as follows Figure 2As shown in the results, the addition of chlorogenic acid and resveratrol alone or in combination to the feed significantly affected the nonspecific immune indicators of Procambarus clarkii serum. Compared with the control group of the basic feed, the activities of lysozyme and phenoloxidase in the serum of Procambarus clarkii in each addition group were significantly increased, and the MDA content was significantly decreased. Compared with the CGA group and the RES group, the lysozyme activity in the serum of Procambarus clarkii in the other groups was significantly increased, and the MDA content was significantly decreased. The lysozyme activity was the highest and the MDA content was the lowest in the CR13 group. The phenoloxidase activity in all mixed addition groups was higher than that in the CGA group and the RES group, and there were significant differences between the CR13 and CR31 groups compared with the CGA group and the RES group. The above results show that the mixed addition of chlorogenic acid and resveratrol in the feed can significantly improve the nonspecific immunity of Procambarus clarkii, and the effect is most obvious when the addition ratio of chlorogenic acid and resveratrol is 1:3.

[0058] 6. Synergistic effects are found

[0059] According to the instructions for use of CompuSyn software, the Combination Index (CI) of chlorogenic acid and resveratrol was calculated to evaluate the synergistic effect between the two. A CI value less than 1 indicates a synergistic effect. The results of the synergistic effect analysis of chlorogenic acid and resveratrol are shown in Table 3:

[0060] Table 3 Analysis of synergistic effect of chlorogenic acid and resveratrol

[0061]

[0062] The results showed that there was a synergistic effect when chlorogenic acid and resveratrol were added in a ratio of 1:3.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of chlorogenic acid combined with resveratrol in the preparation of a product for improving the growth performance and / or disease resistance of Procambarus clarkii, characterized in that: The mass ratio of chlorogenic acid to resveratrol is (1-3):(1-3).

2. A feed additive for improving the growth performance and / or disease resistance of Procambarus clarkii, characterized in that: The feed additive is composed of chlorogenic acid and resveratrol in a mass ratio of (1-3):(1-3).

3. The use of the feed additive according to claim 2 in the culture of Procambarus clarkii, characterized in that: The added amount of the feed additive accounts for 0.04% to 0.16% of the total mass of the feed.

4. The use according to claim 3, characterized in that: The feed additive improves the specific growth rate and survival rate of Procambarus clarkii.

5. The use according to claim 3, characterized in that: The feed additive improves the ability of Procambarus clarkii to resist white spot syndrome virus.

6. The use according to claim 3, characterized in that: The feed additive increases the activities of lysozyme and phenoloxidase in the serum of Procambarus clarkii and reduces the MDA content in the serum of Procambarus clarkii.

7. A feed for Procambarus clarkii, characterized in that: The feed comprises, by mass, 50-150 parts of fish meal, 100-200 parts of soybean meal, 40-80 parts of cottonseed protein, 60-150 parts of rapeseed meal, 50-200 parts of peanut meal, 30-50 parts of corn DDGS, 100-280 parts of wheat flour, 5-20 parts of sodium alginate, 15-25 parts of calcium dihydrogen phosphate, 5-30 parts of yeast powder, 5-30 parts of soybean oil, 5-30 parts of fish oil, 5-15 parts of soybean lecithin oil, 2-8 parts of cholesterol, 5-20 parts of multivitamin, 2-8 parts of sodium chloride, 1-2.5 parts of choline chloride, 1-2.5 parts of vitamin C, 0.5-2 parts of chitosan, 10-50 parts of bentonite, and 0.4-2 parts of feed additives; the feed additives are the feed additives described in claim 2.

8. The crayfish feed according to claim 7, characterized in that: The multivitamin and multiminerals include 5-10 mg / kg thiamine, 5-10 mg / kg riboflavin, 1000-5000 IU / kg vitamin A, 3500-1000 IU / kg vitamin D, and vitamin E. 20-50mg / kg, vitamin K35-10mg / kg, pyridoxine 5-10mg / kg, biotin 0.05-0.2mg / kg, cyanocobalamin 0.02-0.05mg / kg, calcium pantothenate 5-20mg / kg, folic acid 0.5-1mg / kg, niacin 20-50mg / kg, vitamin C 50-200mg / kg, ferrous sulfate 200-400mg / kg, potassium iodate 0.5-1.5mg / kg, cupric chlorohydroxide 2-5mg / kg, zinc sulfate 50-150mg / kg, manganese sulfate 10-50mg / kg, sodium selenite 0.2-0.6mg / kg and cobalt chloride hexahydrate 0.5-1mg / kg.

9. The method for preparing the Procambarus clarkii feed according to any one of claims 7 to 8, characterized in that: The following steps are involved: Fish meal, soybean meal, cottonseed protein, rapeseed meal, peanut meal, corn DDGS, wheat flour, sodium alginate, calcium dihydrogen phosphate, yeast powder, cholesterol, multivitamin, sodium chloride, choline chloride, vitamin C, chitosan, bentonite and feed additives are mixed evenly, and then fish oil, corn oil and soybean lecithin are added, granulated and dried.

10. Use of the Procambarus clarkii feed according to any one of claims 7 to 8 in the preparation of a product that improves the growth performance and / or disease resistance of Procambarus clarkii.

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